Project Summary Plasmodium vivax is the most widely distributed of all of the malaria parasites that cause human disease, and is a source of considerable morbidity, and a major challenge for eradication, due to long-term persistence of asymptomatic hypnozoites in the liver. The biology of P. vivax infections is poorly understood, in large part due to the absence of a system for continuous in vitro culture. P. vivax strikingly invade only reticulocytes during blood-stage infections, the youngest of red blood cells. The molecular mechanisms by which P. vivax invade reticulocytes remain largely unknown. Previous research has focused on a single molecular interaction between a P. vivax invasion ligand PvDBP and the host DARC receptor, but it is now known that invasion can occur through pathways independent of DARC. We hypothesize that other members of the EBL and RBL families of invasion ligands are likely to play a key role in binding to specific reticulocyte receptors to mediate successful invasion. In this proposal, we build upon two major advances in our laboratory- the ability to generate gene knockouts in red blood cells, and the ability to perform robust in vitro invasion assays with P. vivax. Using these approaches, we have identified TfR1 as the receptor for the RBL protein PvRBP2b. We will now 1) conduct a genetic knockout screen of red blood cell membrane proteins to comprehensively identify all of the reticulocyte membrane proteins that are required for P. vivax invasion, and 2) identify key parasite ligands that bind to their cognate red blood cell receptors, by interrogating candidate members of the EBL and RBL families in P. vivax, as well as conducting an unbiased screen of P. vivax merozoite proteins. Together, these studies will serve to dramatically increase our understanding of the molecular interactions between P. vivax and its host red blood cell. In the long-term we hope that our studies will provide a functional understanding of the essential ligand- receptor interactions required for P. vivax invasion, and inform vaccine development and the design of host- targeted therapeutics.